The present invention pertains to block copolymer of the formula: EQU R.sup.1 --L.sup.1 --{R.sup.2 --L.sup.2 --M}.sub.w --L.sup.4 --R.sup.4 --L.sup.3 --R.sup.3 IA.
in which:
either (i) R.sup.1 is a monovalent fluorinated hydrocarbon of 2 to 50 carbon atoms and R.sup.2 is a divalent hydrocarbon of 2 to 50 carbon atoms or (ii) R.sup.1 is a monovalent hydrocarbon of 2 to 50 carbon atoms and R.sup.2 is a divalent fluorinated hydrocarbon of 2 to 50 carbon atoms; PA1 R.sup.3 is (i) hydrogen, (ii) a monovalent fluorinated hydrocarbon of 2 to 50 carbon atoms, or (iii) a monovalent hydrocarbon of 2 to 50 carbon atoms; PA1 R.sup.4 is (i) a bond if R.sup.3 is hydrogen; (ii) a divalent hydrocarbon of 2 to 50 carbon atoms if R.sup.3 is a fluorinated hydrocarbon, or (iii) a divalent fluorinated hydrocarbon of 2 to 50 carbon atoms if R.sup.3 is a hydrocarbon; PA1 each of L.sup.1 and L.sup.2, independently of the other, is a linking group; PA1 L.sup.3 and L.sup.4 taken together with R.sup.4, is a bond if R.sup.3 is hydrogen or if R.sup.3 is other than hydrogen each of L.sup.3 and L.sup.4, taken independently is a linking group; PA1 M is a hydrophilic homopolymer or copolymer comprising at least three monomeric units each having the same or different pendant group containing at least atom selected from the group consisting of oxygen and nitrogen; and PA1 w has a value of from 1 to 100. PA1 (i) R.sup.1 is a monovalent fluorinated hydrocarbon of 2 to 50 carbon atoms and R.sup.2 is a divalent hydrocarbon of 2 to 50 carbon atoms or (ii) R.sup.1 is a monovalent hydrocarbon of 2 to 50 carbon atoms and R.sup.2 is a divalent fluorinated hydrocarbon of 2 to 50 carbon atoms; PA1 n has a value of from 3 to 50 or higher; i.e. 3 to about 200; PA1 R.sup.3 is (i) hydrogen, (ii) a monovalent fluorinated hydrocarbon of 2 to 50 carbon atoms, or (iii) a monovalent hydrocarbon of 2 to 50 carbon atoms; PA1 R.sup.4 is (i) a bond if R.sup.3 is hydrogen; (ii) a divalent hydrocarbon of 2 to 50 carbon atoms if R.sup.3 is a fluorinated hydrocarbon, or (iii) a divalent fluorinated hydrocarbon of 2 to 50 carbon atoms if R.sup.3 is a hydrocarbon; PA1 each of L.sup.1 and L.sup.2, independently of the other, is a linking group; and PA1 L.sup.3 and L.sup.4 taken together with R.sup.4, is a bond if R.sup.3 is hydrogen or if R.sup.3 is other than hydrogen each of L.sup.3 and L.sup.4, taken independently is a linking group. PA1 either R.sup.1 is a monovalent hydrocarbon of 2 to 50 carbon atoms; PA1 R.sup.2 is a divalent fluorinated hydrocarbon of 2 to 50 carbon atoms; PA1 (i) R.sup.3 is hydrogen, R.sup.4, L.sup.3, and L.sup.4 taken together are a bond or PA1 (ii) R.sup.3 is a monovalent hydrocarbon of 2 to 50 carbon atoms, R.sup.4 is a divalent fluorinated hydrocarbon of 2 to 50 carbon atoms, and each of L.sup.1 and L.sup.2, independently of the other, is a linking group; PA1 M is a hydrophilic homopolymer or copolymer comprising at least three monomeric units each having the same or different pendant group containing at least atom selected from the group consisting of oxygen and nitrogen; and PA1 w has a value of from 1 to 100. PA1 A. C.sub.11 F.sub.23 --CONH--C.sub.2 H.sub.4 --NHCOO--(C.sub.2 H.sub.4 O).sub.34 --H PA1 B. C.sub.11 F.sub.23 --CONH--C.sub.8 H.sub.16 --NHCOO--(C.sub.2 H.sub.4 O).sub.34 --H. PA1 C. C.sub.11 F.sub.23 --CONH--C.sub.8 H.sub.16 --NHCOO--(C.sub.2 H.sub.4 O).sub.34 CONH--C.sub.8 H.sub.16 --NHCO--C.sub.11 F.sub.23 PA1 D. C.sub.11 H.sub.23 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F .sub.16 --NHC.sub.2 H.sub.4 NHCO--C.sub.11 H.sub.23 PA1 E: C.sub.12 H.sub.25 NHCOC.sub.8 F.sub.16 CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONHC.sub.2 H.sub.4 NHCOC.sub.8 F.sub.16 CONHC.sub.12 H.sub.12 H.sub.25 PA1 F: C.sub.17 H.sub.35 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --NHC.sub.2 H.sub.4 NHCO--C.sub.17 H.sub.35 PA1 G: C.sub.17 H.sub.35 --CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONHC.sub.2 H.sub.4 NHCO--C.sub.17 H.sub.35 PA1 H: C.sub.11 H.sub.23 --CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONHC.sub.2 H.sub.4 NHCO--C.sub.11 H.sub.23
The present invention also pertains to improved formulations for the administration of a pharmaceutical agent containing at least one block copolymer of Formula I.
These block copolymers can have profound effects on the therapeutic profile of the drug. They can, for example, produce an improvement in the therapeutic index of the drug; i.e., either or both of a decrease in side effects and an increase in therapeutic activity. While the mechanism is not know with certainty, it appears the block copolymers result in one or more of an enhancement of transport into cells and biological barriers such as histohematic barriers which separate the target cells from the perfusing blood; a decrease in sequestration of drug in organs of reticuloendothelial system; a decrease drug metabolism; and an increase drug circulation time in the body.
Moreover with antineoplastic agents, an increase in transport of the drug to solid tumors and a reverse multiple drug resistance often can be observed.
The hydrophilic homopolymer or copolymer M will contain at least three monomeric units, each of which unit will have the same or different pendant group. Each pendant group will contain at least one atom selected from the group consisting of oxygen and nitrogen. Representative hydrophilic homopolymers or copolymers include polyethylene oxides, copolymers of ethylene oxide and propylene oxide, polysaccharides, polyacrylamides, polygycerols, polyvinylalcohols, polyvinylpyrrolidones, polyvinylpyridine N-oxides, copolymers of vinylpyridine N-oxide and vinylpyridine, polyoxazolines, and polyacryloylmorpholine.
Preferably M is ##STR1## ##STR2## ##STR3##
in which each of m and j has a value of from 3 to 5000.
One preferred subgroup includes block copolymers of the formula: EQU R.sup.1 --L.sup.1 --R.sup.2 --L.sup.2 --(C.sub.2 H.sub.4 O).sub.n --L.sup.4 --R.sup.4 --L.sup.3 --R.sup.3 IB.
in which:
Within this subgroup, particularly preferred block copolymers are those of the formula: EQU R.sup.1 --L.sup.1 --{R.sup.2 --L.sup.2 --M}.sub.w --L.sup.4 --R.sup.4 --L.sup.3 --R.sup.3 IC.
in which:
These block copolymers can be viewed as compose of at least three units: a fluorocarbon block, a hydrocarbon block, and a polyoxyethylene block. In a first embodiment, in which R.sup.3 is hydrogen and L.sup.3 and L.sup.4 taken together with R.sup.4 are a bond, the polyoxyethylene block constitutes one terminus of the copolymer with one of either the fluorocarbon block or the hydrocarbon block constituting the other terminus (R.sup.1) and the other (R.sup.2) positioned between. This first embodiment may be viewed as encompassing compounds of the formula: EQU C.sub.x F.sub.y H.sub.z --L.sup.1 --C.sub.q H.sub.2q --L.sup.2 --(C.sub.2 H.sub.4 O).sub.n --H IIA.
in which x has a value of from 2 to 50; y has a value of from 1 to 2x+1, preferably x to 2x+1; z has a value of 2x-y+1; q has a value of from 2 to 50; n has a value of from 3 to 200; and each of L.sup.1 and L.sup.2, independently of the other, is a linking group, and compounds of the formula: EQU C.sub.q H.sub.2q+1 L.sup.1 --C.sub.x F.sub.z --L.sup.2 --(C.sub.2 H.sub.4 O).sub.n --H IIB.
in which x, q, n, L.sup.1, and L.sup.2 are as just defined; y has a value of from x to 2x; and z has a value of 2x-y.
In a second embodiment, two fluorocarbon blocks or two hydrocarbon blocks constitute the termini (R.sup.1 and R.sup.3) while the other bracket the central the polyoxyethylene block. This second embodiment may be viewed as encompassing compounds of the formula: EQU C.sub.x F.sub.y H.sub.z --L.sup.1 --C.sub.q H.sub.2q --L.sup.2 --(C.sub.2 H.sub.4 O).sub.n --L.sup.4 --C.sub.q H.sub.2q --L.sup.3 --C.sub.x F.sub.y H.sub.z IIIA.
in which x has a value of from 2 to 50; y has a value of from 1 to 2x+1, preferably from x to 2x+1; z has a value of 2x-y+1; q has a value of from 2 to 50; n has a value of from 3 to 200; and each of L.sup.1, L.sup.2, L.sup.3, and L.sup.4, independently of the other, is a linking group, and compounds of the formula: EQU C.sub.q H.sub.2q+1 --L.sup.1 --C.sub.x F.sub.y H.sub.z --L.sup.2 --(C.sub.2 H.sub.4 O).sub.n --L.sup.4 --C.sub.x F.sub.y H.sub.z --L.sup.3 --C.sub.q H.sub.2q+1 IIB
in which x, q, n, L.sup.1, L.sup.2, L.sup.3, and L.sup.4 are as just defined; y has a value of from x to 2x; and z has a value of 2x-y.
In the fluorocarbon group C.sub.x F.sub.y H.sub.z --, x has a value of from 2 to 50; i.e., there can be from 2 to 50 carbon atoms. The fluorocarbon group will have at least as many fluorine atoms; i.e., there will be at least x fluorine atoms. If not perfluorinated, the remaining valence bonds of the fluorocarbon group will be satisfied with hydrogen atoms.
Differences in properties can be observed depending upon the nature of R.sup.1 and R.sup.2. Block copolymers of Formulas IA, IIA and IIIA in which R.sup.1 is a monovalent fluorinated hydrocarbon and R.sup.2 is a divalent hydrocarbon tend to be very hydrophobic and interaction with cell membranes and tissues is reduced. These copolymers produce very stable micellar forms of drugs which do not interact with non-target tissues in the body, making them useful as micellar microcontainers for delivery of the drug with decreased drug metabolism and liver uptake. In the block copolymers of Formulas IA, IIA and IIIA, q preferably has a value of 6 or more.
Block copolymers of Formulas IB, IIB and IIIB in which R.sup.1 is a monovalent hydrocarbon and R.sup.2 is a divalent fluorinated hydrocarbon are membranetropic, leading to their activity in MDR cells, which is a consequence of interaction of the copolymer and the cells. Mixtures of the block copolymers produce formulations which provide both the micelle-mediated drug delivery and anti-MDR activity and related activity associated with surfactant interactions with cell membranes. The block copolymers of Formula IB, IIB and IIIB are preferred.
The linking groups L.sup.1, L.sup.2, L.sup.3, and L.sup.4 generally do not contribute to the final utility of the compounds but serve to covalently join the blocks of the copolymer chain. Accordingly a wide variety of divalent linking groups can be employed.
Linking can be accomplished by a number of reactions, many of which have been described generally in conjugate chemistry. These can involve a terminal hydroxyl group on a R.sup.5 --O--(C.sub.2 H.sub.4 O)--H block, in which R.sup.5 is hydrogen or a blocking group such as alkyl, and an appropriate group on the C.sub.x F.sub.y H.sub.z -- or C.sub.q H.sub.2q -- block, the two being joined directly or indirectly; i.e., through a third component. Alternatively a terminal group can be converted to some other functional group, as for example amino, which then is allowed to react with either with the next block component or another linking component. The linking group thus may be formed either by reactively involving a terminal group of a block or by replacing the terminal group. For example, a carboxylic acid group can be activated as with N,N'-dicyclohexylcarbodiimide and then allowed to react with an amino or hydroxy group to form an amide or ether respectively. Anhydrides and acid chlorides will produce the same links with amines and alcohols. Alcohols can be activated b y carbonyldiimidazole and then linked to amines to produce urethane linkages. Alkyl halides can be converted to an amines or allowed to react with an amine, diamines, alcohols, or diol. A terminal hydroxy group of the R.sup.5 --O--(C.sub.2 H.sub.4 O)--H block can be oxidized to form the corresponding aldehyde or ketone. This aldehyde or ketone then is allowed to react with a precursor carrying a terminal amino group to form an imine which, in turn, is reduced, as with sodium borohydrate to form the secondary amine. See Kabanov et al., J. Controlled Release, 22:141 (1992); Meth. Enzymol., XLVII, Hirs & Timasheff, Eds., Acad. Press, 1977. The linkage thereby formed is the group --NH--, replacing the terminal hydroxyl group of the R.sup.5 --O--(C.sub.2 H.sub.4 O)--H block.
Alternatively, a terminal hydroxyl group on the R.sup.5 --O--(C.sub.2 H.sub.4 O)--H polymer can be allowed to react with bromoacetyl chloride to form a bromoacetyl ester which in turn is allowed to react with an amine precursor to form the --NH--CH.sub.2 --C(O)-- linkage. Immobilized Enzymes, Berezin et al., Eds., MGU, Moscow, 1976, i.e., --NH--CH.sub.2 --C(O)--.
The bromoacetyl ester of a R.sup.1 --R.sup.2 --R.sup.3 --H polymer, prepared as described above, also can be allowed to react with a diaminoalkane of the formula NH.sub.2 --C.sub.q H.sub.2q--NH.sub.2 which in turn is allowed to react with a acid precursor of the formula C.sub.x F.sub.y H.sub.z --COOH, or an activated derivative thereof such as an acid chloride or anhydride, to yield a compound of the formula: EQU C.sub.x F.sub.y H.sub.z --CO--NH--C.sub.q H.sub.2q --NHCH.sub.2 COO--R.sup.1 --R.sup.2 --R.sup.3 --H. IV
The bromoacetyl ester also can be allowed to react with a cyanide salt to form a cyano intermediate. See, e.g., Sekiguchi et al., J. Biochem., 85, 75 (1979); Tuengler et al., Biochem. Biophys. Acta, 484, 1(1977); Browne et al, BBRC, 67 126 (1975); and Hunter et al., J.A.C.S., 84, 3491(1962). This cyano intermediate then can be converted to an imido ester, for instance by treatment with a solution of methanol and hydrogen chloride, which is allowed to react ed with a amine precursor to form a --NH--C(NH.sub.2.sup.+)CH.sub.2 C(O)-- linkage.
A terminal hydroxyl group also can be allowed to react with 1,1'-carbonyl-bis-imidazole and this intermediate in turn allowed to react with an amino precursor to form a --N--C(O)O-- linkage. See Bartling et al., Nature, 243:342 (1973).
A terminal hydroxyl also can be allowed to react with a cyclic anhydride such as succinic anhydride to yield a half-ester which, in turn, is allowed to react with a precursor of the formula C.sub.x F.sub.y H.sub.z --NH.sub.2 using conventional condensation techniques for forming peptide bonds such as dicyclohexylcarbodiimide, diphenylchlorophosphonate, or 2-chloro-4,6-dimethoxy-1,3,5-triazine. See e.g., Means et al., Chemical Modification of Proteins, Holden-Day (1971). Thus formed is the --NHC(O)(CH.sub.2).sub.q C(O)O-- linkage.
A terminal hydroxyl group also can be allowed to react with 1,4-butanediol diglycidyl ether to form an intermediate having a terminal epoxide function linked to the polymer through an ether bond. The terminal epoxide function, in turn, is allowed to react with an amino precursor. Pitha et al., Eur. J. Biochem., 94:11 (1979); Elling and Kula, Biotech. Appl. Biochem., 13:354 (1991); Stark and Holmberg, Biotech. Bioeng., 34:942 (1989).
Halogenation of a terminal hydroxyl group permits subsequent reaction with an alkanediamine such as 1,6-hexanediamine. The resulting product then is allowed to react with carbon disulfide in the presence of potassium hydroxide, followed by the addition of proprionyl chloride to generate a isothiocyanate which in turn is allowed to react with an amino precursor to yield a --N--C(S)--N--(CH.sub.2).sub.6 --NH-- linkage. See Means et al., Chemical Modification of Proteins, Holden-Day (1971).
The R.sup.1 --R.sup.2 --R.sup.3 --H polymer chain terminating in an amino group also can be treated with phosgene and then with a precursor of the formula C.sub.x F.sub.y H.sub.z --NH.sub.2 to form a urea linkage. See Means et al., Chemical Modification of Proteins, Holden-Day (1971).
The block precursor terminating in an amino group also can be treated with dimethyl ester of an alkane dicarboxylic acid and the product allowed to react with an amino precursor to produce a --N--C(NH.sub.2.sup.+)--(CH.sub.2).sub.4 --C(NH.sub.2.sup.+)--N- linkage. See Lowe et al., Affinity Chromatography, Wiley & Sons (1974).
The block precursor terminating in an amino group also can be allowed to react with an alkanoic acid or fluorinated alkanoic acid, preferably an activated derivative thereof such as an acid chloride or anhydride, to form a linking group --CONH--.
Alternatively an amino precursor can be treated with an .alpha.,.omega.-diisocyanoalkane to produce a --NC(O)NH(CH.sub.2).sub.6 NHC(O)--N-- linkage. See Means, Chemical Modification of Proteins, Holden-Day (1971).
Some linking groups thus can simply involve a simple functional group while others may comprise a spacer unit such as a polymethylene chain between two functional groups. When the linking group comprises such a polymethylene chain, it can have as few as two methylene units but preferably contains more; e.g., six or more methylene units.
The above descriptions exemplify typical strategies for the formation of linkages between the blocks of the copolymers. These procedures parallel those which are known to form conjugates of biologically active agents and other agents. As but one example, a poly(ethylene glycol) of the formula R.sup.5 --O--(C.sub.2 H.sub.4 O)--H in which R.sup.5 is hydrogen and a diamine of the formula H.sub.2 N--R.sup.2 --NH.sub.2 can be coupled utilizing 1,1'-carbonyldimidazole. This intermediate is then allowed to react with an alkanoic acid or fluorinated alkanoic acid of the formula R.sup.1 COOH which has been activated with N,N'-dicyclohexylcarbodiimide. The resultant block copolymers can be separated by conventional chromatography; e.g., using silica gel column. Other strategies can be used, including the general conjugation methods described by Means et al., Chemical Modification of Proteins, Holden-Day (1971); Glazer et al., Chemical Modification of Proteins, Elsevier, N.Y. (1975); Immunotechnology Catalog & Handbook, Pierce Chemical Co.; and Polyethylene Glycol DerivativesCatalog, Shearwater Polymers, Inc. (1994).
It also will be appreciated that linkages which are not symmetrical, such as --CONH-- or --NHCOO--, can be present in the reverse orientation; e.g., --NHCO-- and --OCONH--, respectively.
Suitable starting materials for block copolymers in which R.sup.1 is a monovalent fluorinated hydrocarbon include fluoroalkylcarboxylic acids, anhydrides and acid chlorides of fluoroalkylcarboxylic acids such as monofluoroacetic acid, trifluoroacetic acid, pentafluoropropionic acid, heptafluorobutyric acid, heptafluorobutyric anhydride, heptafluorobutyrylchloride, nonafluoropentanoic acid, tridecafluoroheptanoic acid, pentadecafluorooctanoic acid, heptadecafluorononanoic acid, nonadecafluorodecanoic acid, perfluorododecanoic acid, and perfluorotetradecanoic acid; fluoroalkanols such as 2,2,3,3,4,4,4-heptafluoro-1-butanol, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluoro-1-octanol, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,9-heptadecafluoro-1-nonanol, 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-nonadecafluoro-1-decanol, and 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heneicosafluoro-1-undecanol ; fluoroalkyl halides such as perfluoroethyl iodide, perfluoropropyl iodide, perfluorohexyl bromide, perfluoroheptyl bromide, perfluorooctyl bromide, perfluorooctyl iodide, 1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8-heptadecafluoro-10-iododecane, perfluorodecyl iodide, perfluorododecyl iodide, and 1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8-heptadecafluoro-10-iododecane; fluoroalkylamines, and fluoroalkylaldehydes; and the like.
Suitable starting materials for block copolymers in which R.sup.1 is a monovalent hydrocarbon include carboxylic acids, anhydrides and acid chlorides of carboxylic acids such as valeric acid, valeric anhydride, 2,4-pentadienoic acid, hexanoic acid, hexanoic anhydride, hexanoyl chloride, 2-hexenoic acid, 3-hexenoic acid, 2,6-heptadienoic acid, 6-heptenoic acid, heptanoic acid, 2-octenoic acid, octanoic acid, octanoyl chloride, nonanoic acid, nonanoyl chloride, decanoic acid, decanoic anhydride, decanoyl chloride, undecanoic acid, undecanoic anhydride, undecanoyl chloride, undecelynic acid, 10-undecenoyl chloride, lauric acid, lauric anhydride, lauroyl chloride, myristoleic acid, myristic acid, myristic anhydride, myristoyl chloride, palmitic acid, palmitic anhydride, palmitoyl chloride, palmitoleic acid, heptadecanoic acid, oleic acid, oleic anhydride, oleoyl chloride, stearic acid, stearic anhydride, stearoyl chloride, nonanedecanoic acid, arachidonic acid, heneicosanoic acid, docasanoic acid, docasanoic anhydride, tricosanoic acid, tetracosanoic acid, tetracos-15-enoic acid, hexacosanoic acid, heptacosanoic acid, octacosanoic acid, triocantanoic acid, and the like; alkanols such as heptanol, octanol, nonanol, decanol, undecanol, undecyl-9-en-1-ol, dodecanol, 1-tetradecanol, hexadecanol, hexadec-11-en-1-ol, heptadecanol, oleyl alcohol, octadecanol, nonanedecanol, hexacosanol, 1-triocantanol, and the like; aldehydes such as heptaldehyde, octyl aldehyde, decyl aldehyde, undecylic aldehyde, undecylenic aldehyde, dodecyl aldehyde, tetradecyl aldehyde, oleic anhydride and the like; and alkyl amines such as hexylamine, heptylamine, octylamine, decylamine, undecylamine, dodecylamine, pentadecyl amine, hexadecyl amine, oleylamine, stearylamine, and the like
Suitable starting materials for block copolymers in which R.sup.2 is a divalent fluorinated hydrocarbon include fluorodicarboxylic acids, and anhydrides and acid chlorides of fluorodicarboxylic acids such as tetrafluorosuccinic acid, hexafluoroglutaric acid, hexafluoroglutaric anhydride, perfluoroadipic acid, perfluorosuberic acid, perfluorosebacic acid, and the like; and fluorinated alkanediols such as 2,2,3,3,4,4,5,5-octa-fluoro-1,6-hexanediol and 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluoro-1,10-decanediol, and the like.
Suitable starting materials for block copolymers in which R.sup.2 is a divalent hydrocarbon include dicarboxylic acids and anhydrides and acid chlorides of dicarboxylic acids such as succinic acid, pimelic acid, pimeloyl chloride, suberic acid, sebacicic acid, sebacyl chloride, azelaic acid, azelaoyl chloride, undecanedioic acid, 1,10-decanedicarboxylic acid, dodecanedioyl chloride, 1,11-undecanedicarboxylic acid, 1,12-dodecanedicarboxylic acid, 1,12-dodecanedioyl dichloride, hexadecanedioic acid, docosanedioic acid, tetracosanedioic acid, and the like; alkanediols such as 1,10-decanediol, 1,12-dodecanediol, 1,16-hexadecanediol, and the like; and diamines such as 1,2-diaminoethane, 1,3-diaminopropane, 1,4-diaminobutane, 1,5-diaminopentane, 1,6-diaminohexane, 1,7-diaminoheptane, 1,8-diaminooctane, 1,9-diaminononane, 1,10-diaminodecane, 1,12-diaminododecane, and the like; and dialdehydes and semi-aldhydes such as succinic dialdehyde, and succinic semialdehyde, and the like.
The hydrophilic homopolymers or copolymers encompassed by M include polyethylene oxides, copolymers of ethylene oxide and propylene oxide, polysaccharides, polyacrylamides, polygycerols, polyvinylalcohols, polyvinylpyrrolidones, polyvinylpyridine N-oxides, copolymers of vinylpyridine N-oxide and vinylpyridine, polyoxazolines, and polyacroylmorpholines. These polymers are commercially available and can be synthesized as functionally terminated derivatives for subsequent conjugation by polymerization of respective monomers as described by Veronese et al., J. Bioact. Comp. Polym. 5:167 (1990); Sartore et al., J. Bioact. Compat. Polym. 9:411 (1994); Ranucci et al., Macromol. Chem. Phys. 195:3469 (1994); Torchilin et al., Biochim. Biophys. Acta. 1195:181 (1994); Torchilin et al., J. Pharm. Sci. 84:1049 (1995).
By way of example, a large number of monofunctional and difunctional poly(ethylene glycol) starting materials are available. Poly(ethylene glycol) with average molecular weights of from a few hundred to 50,000, as well as monomethoxy derivatives thereof, are commercially available; e.g., mw of 200, 1,000, 5,000, 10,000, and 25,000.
The block co-polymers of the present invention are utilized in pharmaceutical compositions intended for oral, topical (including optical and transdermal as through a topical patch), rectal, vaginal, pulmonary, or parenteral (such as intramuscular, subcutaneous, intraperitoneal or intravenous) administration. These pharmaceutical compositions can take the form of tablets, capsules, lozenges, troches, powders, gels, syrups, elixirs, aqueous solutions, suspensions, micelles, emulsions, and microemulsions.
Conventional pharmaceutical formulations are employed. In the case of tablets, for example, well-known carriers such as lactose, sodium citrate, and salts of phosphoric acid can be used. Disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, as are commonly used in tablets, can be present. Capsules for oral administration can include diluents such as lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the conjugate can be combined with emulsifying and suspending agents. For parenteral administration, sterile solutions of the conjugate are usually prepared, and the pH of the solutions are suitably adjusted and buffered. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic. For ocular administration, ointments or droppable liquids may be delivered by well-known ocular delivery systems such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or polyvinyl alcohol, preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers. For pulmonary administration, diluents and/or carriers will be selected to be appropriate to allow the formation of an aerosol.
The pharmaceuticals with which the present block copolymers can be an agent that is useful for diagnostics or imaging or an agent that acts upon a cell, organ, or organism to create a change in the functioning of the cell, organ or organism. They thus include but are not limited to pharmaceutical drugs, immunoadjuvants, vaccines, and the like. Pharmaceutical agents are represented by wide variety of agents that are used in diagnostics, therapy, immunization or otherwise are applied to combat human and animal disease such as nucleic acids, polynucleotides, antibacterial agents, antiviral agents, antifungal agents, antiparasitic agents, tumoricidal or anti-cancer agents, proteins, toxins, enzymes, hormones, neurotransmitters, glycoproteins, immunoglobulins, immunomodulators, dyes, radiolabels, radio-opaque compounds, fluorescent compounds, polysaccharides, cell receptor binding molecules, anti-inflammatories, anti-glaucomic agents, mydriatic compounds, local anesthetics, DNA topoisomerase inhibitors (including type I and type II), brain and tumor imaging agents, free radical scavenger drugs, anticoagulants, ionotropic drugs, and neuropeptides such as endorphins.
Thus the pharmaceutical agents include non-steroidal anti-inflammatories such as indomethacin, salicylic acid acetate, ibuprofen, sulindac, piroxicam, and naproxen, antiglaucomic agents such as timolol or pilocarpine, neurotransmitters such as acetylcholine, anesthetics such as dibucaine, neuroleptics such as the phenothiazines (for example compazine, thorazine, promazine, chlorpromazine, acepromazine, aminopromazine, perazine, prochlorperazine, trifluoperazine, and thioproperazine), rauwolfia alkaloids (for example, reserpine and deserpine), thioxanthenes (for example chlorprothixene and tiotixene), butyrophenones (for example haloperidol, moperone, trifluoperidol, timiperone, and droperidol), diphenylbutylpiperidines (for example pimozide), and benzamides (for example sulpiride and tiapride); tranquilizers such as glycerol derivatives (for example mephenesin and methocarbamol), propanediols (for example meprobamate), diphenylmethane derivatives (for example orphenadrine, benzotrapine, and hydroxyzine), and benzodiazepines (for example chlordiazepoxide and diazepam); hypnotics (for example zolpdem and butoctamide); beta-blockers (for example propranolol, acebutonol, metoprolol, and pindolol); anti-depressants such as dibenzazepines (for example, imipramine), dibenzocycloheptenes (for example, amitriptyline), and the tetracyclics (for example, mianserine); MAO inhibitors (for example phenelzine, iproniazid, and selegeline); psychostimulants such as phenylethylamine derivatives (for example amphetamines, dexamphetamines, fenproporex, phentermine, amfeprramone, and pemoline) and dimethylaminoethanols (for example clofenciclan, cyprodenate, aminorex, and mazindol); GABA-mimetics (for example, progabide); alkaloids (for example codergocrine, dihydroergocristine, and vincamine); anti-Parkinsonism agents utilized in (for example L-dopamine and selegeline); agents utilized in the treatment of Alzheimer's disease, cholinergics (for example citicoline and physostigmine); vasodilators (for example pentoxifyline); and cerebro active agents (for example pyritinol and meclofenoxate).
The block copolymers also can be used advantageously with anti-neoplastic agents such as paclitaxel, daunorubicin, doxorubicin, carminomycin, 4'-epiadriamycin, 4-demethoxy-daunomycin, 11-deoxydaunorubicin, 13-deoxydaunorubicin, adriamycin-14-benzoate, adriamycin-14-octanoate, adriamycin-14-naphthaleneacetate, vinblastine, vincristine, mitomycin C, N-methyl mitomycin C, bleomycin A.sub.2, dideazatetrahydrofolic acid, aminopterin, methotrexate, cholchicine and cisplatin, antibacterial agents such as aminoglycosides including gentamicin, antiviral compounds such as rifampicin, 3'-azido-3'-deoxythymidine (AZT), and acylovir; antifungal agents such as azoles including fluconazole, macrolides such as amphotericin B, and candicidin; anti-parasitic compounds such as antimonials.
The compositions also can utilize a variety of polypeptides such as antibodies, toxins such as diphtheria toxin, peptide hormones, such as colony stimulating factor, and tumor necrosis factors, neuropeptides, growth hormone, erythropoietin, and thyroid hormone, lipoproteins such as .alpha.-lipoprotein, proteoglycans such as hyaluronic acid, glycoproteins such as gonadotropin hormone, immunomodulators or cytokines such as the interferons or interleukins, hormone receptors such as the estrogen receptor.
The block copolymers also can be used with enzyme inhibiting agents such as reverse transcriptase inhibitors, protease inhibitors, angiotensin converting enzymes, 5.alpha.-reductase, and the like. Typical of these agents are peptide and nonpeptide structures such as finasteride, quinapril, ramipril, lisinopril, saquinavir, ritonavir, indinavir, nelfinavir, zidovudine, zalcitabine, allophenylnorstatine, kynostatin, delaviridine, bis-tetrahydrofuran ligands (see, for example Ghosh et al., J. Med. Chem. 39(17): 3278-90 1966), and didanosine. Such agents can be administered alone or in combination therapy; e.g., a combination therapy utilizing saquinavir, zalcitabine, and didanosine or saquinavir, zalcitabine, and zidovudine. See, for example, Collier et al., Antiviral Res., 1996 Jam. 29(1): 99.
The block copolymers also can be used with nucleic acids such as thymine, polynucleotides such as DNA or RNA polymers or synthetic oligonucleotides, which may be derivatized by covalently modifying the 5' or the 3' end of the polynucleic acid molecule with hydrophobic substituents to facilitate entry into cells. These modified nucleic acids generally gain access to the cells interior with greater efficiency. See, for example, Kabanov et al., FEBS Lett., 259:327 (1990); Boutorin et al., FEBS Lett., 23:1382-1390, 1989; Shea et al, Nucleic Acids Res., 18:3777-3783, 1990. Additionally, the phosphate backbone of the polynucleotides has been modified to remove the negative charge (see, for example, Agris et al., Biochemistry, 25:6268 (1986); Cazenave and Helene in Antisense Nucleic Acids and Proteins: Fundamentals and Applications, Mol and Van der Krol, Eds., p. 47 et seq., Marcel Dekker, New York, 1991) or the purine or pyrimidine bases have been modified (see, for example, Antisense Nucleic Acids and Proteins: Fundamentals and Applications, Mol and Van der Krol, Eds., p. 47 et seq., Marcel Dekker, New York, 1991; Milligan et al. in Gene Therapy For Neoplastic Diseases, Huber and Laso, Eds., p. 228 et seq., New York Academy of Sciences, New York, 1994). Such nucleic acid molecules can be among other things antisense nucleic acid molecules, phosphodiester, oligonucleotide .alpha.-anomers, ethyl phospotriester analogs, phosphorothioates, phosphorodithioates, phosphoroethyletriesters, methylphosphonates, and the like (see, for example, Crooke, Anti-Cancer Drug Design 1991, 6:609; De Mesmaeker et al., Acc. Chem. Res. 1995, 28: 366). The invention is used with antigene, ribozyme and aptamer nucleic acid drugs (see, for example, Stull and Szoka, Pharm. Res. 1995, 12:465).
Included among the suitable pharmaceutical agents are viral genomes and viruses (including the lipid and protein coat). This accounts for the possibility of using our invention with a variety of viral vectors in gene delivery; e.g., retroviruses, adenoviruses, herpes virus, pox virus, used as complete viruses of their parts. See, for example, Hodgson, Biotechnology, 1995, 13: 222.
The suitable pharmaceutical agents include oxygen transporters (e.g. porphines, porphirines and their complexes with metal ions), coenzymes and vitamins (e.g. NAD/NADH, vitamins B12, chlorophylls), and the like.
The suitable pharmaceutical agents further include the agents used in diagnostics visualization methods, such as magnetic resonance imaging (e.g., gadolinium (III) diethylenetriamine pentaacetic acid), and may be a chelating group (e.g., diethylenetriamine pentaacetic acid, triethylenetriamine pentaacetic acid, ethylenediamine-tetraacetic acid, 1,2-diaminocyclohexane-N,N,N',N'-tetraaceticacid, N,N'-di-(2-hydroxybenzyl)ethylene diamine), N-(2-hydroxyethyl)ethylene diamine triacetic acid and the like). Such pharmaceutical agent may further include an alpha-, beta-, or gamma-emitting radionuclide (e.g., gallium 67, indium 111, technetium 99). The suitable pharmaceutical agents also include iodine-containing radiopaque molecules. The pharmaceutical agent may also be a diagnostic agent, which may include a paramagnetic or superparamagnetic element, or combination of paramagnetic element and radionuclide. The paramagnetic elements include but are not limited to gadolinium (III), dysporsium (III), holmium (III), europium (III) iron (III) or manganese (II).
The pharmaceutical adjuncts of this invention can be also used in fibrinolitic compositions with enzymes such as streptokinase, urokinase, tissue plasminogen activator or other fibrinolitic enzyme that is effective in dissolving blood clots and reestablishing and maintaining blood flow through thrombosed coronary or other blood vessels. Also these pharmaceutical adjuncts are used in compositions for treating burns, circulatory diseases in which there is an acute impairment of circulation, in particular, microcirculation, respiratory distress syndrome, as well as compositions for reducing tissue damage during angioplasty procedures. Further, the compositions of the pharmaceutical adjuncts including but not limited to aqueous solutions of the effective concentrations of these adjuncts are used to treat myocardial damage, ischemic tissue, tissue damaged by reperfusion injury, stroke, sickle cell anemia and hypothermia. These compositions are especially useful for treating vascular obstructions caused by abnormal cells which is an often complication during malaria and leukemia and are suitable as a perfusion medium for transplantation of organs. The pharmaceutical adjuncts of this invention are also suitable for use in compositions as antiinfective compounds, as well as modulators of immune response, and improved adjuvants, antigenes and vaccines.
The adjuvants suitable for use with the pharmaceutical adjuncts of this invention include but are not limited to adjuvants of mineral, bacterial, plant, synthetic or host product origin. The suitable mineral adjuvants include aluminum compounds such as aluminum particles and aluminum hydroxide. The suitable bacterial adjuvants include but are not limited to muramyl dipeptides, lipid A, Bordetella pertussis, Freund's Complete Adjuvant, lipopolysaccharides and its various derivatives, and the like. The suitable adjuvants include without limitation small immunogenes, such as synthetic peptide of malaria, polysaccharides, proteins, bacteria and viruses. The antigenes that can be used with the pharmaceutical adjuncts of the present invention are compounds which, when introduced into a mammal will result in formation of antibodies. The suitable antigens include but are not limited to natural, recombinant, or synthetic products derived from viruses, bacteria, fungi, parasites and other infectious agents, as well as autoimmune disease, hormones or tumor antigens used in prophylactic or therapeutic vaccines. These antigens include components produced by enzymatic cleavage or can be compounds produced by recombinant DNA technique. Viral antigens include but are not limited to HIV, rotavirus, influenza, foot and mouth disease, herpes simplex, Epstein Barr virus, Chicken pox, pseudorabies, rabies, hepatitis A, hepatitis B, hepatitis C, measles, distemper, Venezuelan equine encephalomyelitis, Rota virus, polyoma tumor virus, Feline leukemia virus, reovirus, respiratory synticial virus, Lassa fever virus, canine parvovirus, bovine pappiloma virus, tick borne encephalitis, rinderpest, human rhinovirus species, enterovirus species, Mengo virus, paramixovirus, avian infectious bronchitis virus. Suitable bacterial antigens include but are not limited to Bordetella pertussis, Brucella abortis, Escherichia coli, salmonella species, salmonella typhi, streptococci, cholera, shigella, pseudomonas, tuberculosis, leprosy and the like. Also suitable antigens include infections such as Rocky mountain spotted fever and typhus, parasites such as malaria, schstosomes and trypanosomes, and fungus such as Cryptococcus neoformans. The protein and peptide antigens include subunits of recombinant proteins (such as herpes simplex, Epstein Barr virus, hepatitis B, pseudorabies, flavivirus, Denge, yellow fever, Neissera gonorrhoeae, malaria, trypanosome surface antigen, alphavirus, adenovirus and the like), proteins (such as diphtheria toxoid, tetanus toxoid, meningococcal outer membrane protein, streptococcal M protein, hepatitis B, influenza hemagglutinin and the like), synthetic peptides (e.g. malaria, influenza, foot and mouth disease virus, hepatitis B, hepatitis C). Suitable polysaccharide and oligosaccharide antigens originate from Haemphilis influenza, Neisseria meningitides, Pseudomonas aeruginosa, Klebsiella pneumoniae, pneumococcus.
Preferred classes of biological agents include anti-neoplastic agents, antibacterial agents, antiparasitic agents, CNS agents, immunomodulators and cytokines, toxins, neuropeptides and polynucleotides. Biological agents, such as certain drugs for which target cells tend to develop resistance mechanisms are also preferred. Particularly preferred biological agents include anthracyclines such as doxorubicin, daunorubicin, or carminomycin, vinca alkaloids, mitomycin-type antibiotics, bleomycin-type antibiotics, flucanazol, amphotericin B, paclitaxel and derivatives, immunomodulators and cytokines such as interleukins and TNFS, erythropoietin, and polynucleotides, especially oligonucleotides.
The ability of the present block copolymers to alter the biological profile and activity of pharmaceutical agents can be conveniently observed in a number of experimental models. The block copolymers for example can affect the uptake of antineoplastic agents in multidrug resistant cancer cells. The multidrug resistant KBv cell line (vinblastine resistant human epidermoid carcinoma) which expresses high levels of glycoprotein P (P-gp) efflux pump (Gervasoni, et al. Cancer Research, 1991, 51, 4955) can be used to evaluate the effects of the block copolymers on rhodamine 123. Rhodamine 123 is a specific probe for the effects on the P-gp efflux system, which is commonly used for evaluation of the P-gp efflux function in cancer and normal cells (Jancis, et al, Mol. Pharmacol. 1993, 43, 51; Lee, et al., Mol. Pharmacol. 1994, 46, 627). The results with rhodamine 123 are indicative of the effects on the transport of all MDR class drugs, including anthracycline antibiotics. To maintain the high expression of P-gp in the KBv cells, the cells are cultured in DMEM supplemented with 10% FBS and 1 mg/ml vinblastine. The KBv cell monolayers are grown in 24-well culture plates and used in rhodamine 123 uptake experiments after reaching confluency. Confluency of all the cell monolayers is determined by visual inspection using an inverted light microscope. The uptake of rhodamine 123 in KBv cell monolayers in presence and absence of the block copolymers is examined at 37.degree. C. over a period of 90 minutes. The culture media is removed from the KBv monolayers and replaced with an assay buffer having the following composition: NaCl (122 mM), NaHCO.sub.3 (25 mM), glucose (10 mM), KCl (3 mM), MgSO.sub.4 (1.2 mM), K.sub.2 HPO.sub.4 (0.4 mM), CaCl.sub.2 (1.4 mM) and HEPES (10 mM). After a thirty-minute pre-incubation at 37.degree. C., the assay buffer is removed from the monolayers and 3.2 .mu.M rhodamine in the assay buffer or 3.2 .mu.M rhodamine solubilized in 0.1% (wt.) of the block copolymer are added to the monolayers. These samples are exposed to the monolayers at 37.degree. C. for 90 minutes and uptake then stopped by removing the medium and washing the KBv monolayers three times with 0.5 ml ice-cold PBS. The KBv monolayers are solubilized in 1.0% Triton X-100 (0.5 ml) and aliquots are removed for determining cell-associated rhodamine fluorescence and protein content. Rhodamine 123 fluorescence is determined at .lambda..sub.ex =488 nm and .lambda..sub.em =550 nm using Shimadzu 5000 spectrophotometer; and protein content is determined using the Pierce BCA method. The concentration of rhodamine in the KBv lyzate solution can be quantitatively determined by construction of standard curves. Data from the uptake studies are expressed as amount of cell-associated rhodamine/mg protein. Each data point represents the mean.+-.SEM of 4 monolayers.
For the block copolymer C.sub.11 F.sub.23 --CONH--C.sub.2 H.sub.4 --NHCOO--(C.sub.2 H.sub.4 O).sub.34 --H, the results are as follows:
Cell-associated rhodamine, Composition studied nmole/mg protein Rhodamine in assay buffer 0.26 .+-. 0.01 Rhodamine/block copolymer 0.32 .+-. 0.02
For the block copolymer C.sub.11 F.sub.23 --CONH--C.sub.2 H.sub.4 --NHCOO--(C.sub.2 H.sub.4 O).sub.9-10 --H, the results are as follows:
 Cell-associated rhodamine, Composition studied nmole/mg protein Rhodamine in assay buffer 0.26 .+-. 0.01 Rhodamine/block copolymer 0.34 .+-. 0.02
For the copolymer C.sub.11 F.sub.23 --CONH--C.sub.2 H.sub.4 --NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONH--C.sub.2 H.sub.4 NHCO--C.sub.11 F.sub.23, the results are as follows:
 Cell-associated rhodamine, Composition studied nmole/mg protein Rhodamine in assay buffer 0.63 .+-. 0.04 Rhodamine/block copolymer 0.60 .+-. 0.02
For the copolymer:
C.sub.11 H.sub.23 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --NHC.sub.2 H.sub.4 NHCO--C.sub.11 H.sub.23 PA0 A: C.sub.11 H.sub.23 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --H PA0 B: CH.sub.11 H.sub.23 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --NHC.sub.2 H.sub.4 NHCO--C.sub.11 H.sub.23 PA0 C: C.sub.17 H.sub.35 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O ).sub.34 --CONHC.sub.2 H.sub.4 NHCO--C.sub.8 F.sub.16 --NHC.sub.2 H.sub.4 NHCO--C.sub.17 H.sub.35 PA0 D: C.sub.12 H.sub.25 NHCOC.sub.8 F.sub.16 CONHC.sub.2 H.sub.4 NHCOO--(C.sub.2 H.sub.4 O).sub.34 --CONHC.sub.2 H.sub.4 NHCOC.sub.8 F.sub.16 CONHC.sub.12 H.sub.25
the results are as follows:
 Cell-associated rhodamine, Composition studied nmole/mg protein Rhodamine in assay buffer 0.54 .+-. 0.04 Rhodamine/block copolymer 2.80 .+-. 0.14
P-gp also controls the permeability of human intestinal epithelial cells (Caco-2) with respect to numerous pharmaceutical agents (Thiebault et al. Proc. Natl. Acad. Sci. USA 1987, 84: 7735) and surfactants such as Cremophor EL can significantly enhance the permeability of Caco-2 cells to selected peptides (Nerurkar et al., Pharm. Res., 1996, 13: 528), presumably through inhibition of drug efflux transport systems in these cells.
The effects of the fluorinated copolymers on rhodamine 123 uptake can be observed following the procedure described above, modified however by replacing KBv cells monolayers with Caco-2 cells monolayers.
The following fluorinated block copolymers were used:
The control employed 10 .mu.g/mL cyclosporin A (CSA), an inhibitor of P-gp on the uptake of rhodamine 123. The results are as follows:
 Cell associated rhodamine 123, Composition studied nmol/mg Rhodamine 123 in assay buffer 0.54 .+-. 0.04 Rhodamine 123 in CSA 3.02 .+-. 0.11 Rhodamine 123 in 0.1% Copolymer A 0.38 .+-. 0.03 Rhodamine 123 in 0.1% Copolymer B 2.81 .+-. 0.14 Rhodamine 123 in 0.05% Copolymer B 4.68 .+-. 0.29 Rhodamine 123 in 0.025% Copolymer B 5.31 .+-. 0.04 Rhodamine 123 in 0.1% Copolymer C 1.77 .+-. 0.14 Rhodamine 123 in 0.01% Copolymer C 2.51 .+-. 0.25 Rhodamine 123 in 0.001% Copolymer C 1.64 .+-. 0.06 Rhodamine 123 in 0.125% Copolymer D 2.33 .+-. 0.21
The block copolymers also have an effect on drug uptake across the blood brain barrier as can be demonstrated in the following model. Bovine blood brain microvessel endothelial cells (BBMEC) are isolated from fresh cow brains using enzymatic digestion and density centrifugation as described by Miller et al. (J. Tissue Cult. Meth., 1992, 14, 217). The BBMEC are seeded at density of 50000 cells/cm.sup.2 onto collagen-coated, fibronectin-treated 24-well culture plates in media consisting of 45% minimum essential medium (MEM), 45% Ham's F-12 (F12), and 10% horse serum, supplemented with antibiotics and heparin sulfate as described in Miller et al., supra. The media is will be replaced every other day with fresh media and studies are performed on confluent BBMEC monolayers (10-12 days). The uptake studies using rhodamine 123 in assay buffer and rhodamine 123 (3.2 .mu.M) and rhodamine 123 (3.2 .mu.M) solubilized in 0.01% (wt.) of block copolymer are performed as described supra.
For the block copolymer C.sub.11 F.sub.23 --CONH--C.sub.2 H.sub.4 --NHCOO--(C.sub.2 H.sub.4 O).sub.34 --H, the results are as follows:
 Cell associated rhodamine, Composition studied nmol/mg Rhodamine in assay buffer 1.61 .+-. 0.25 Rhodamine/block copolymer 2.13 .+-. 0.13
The above data demonstrate that the block copolymers enhance transport of MDR drug into cells expressing P-gp efflux system and that formulation with the block copolymers increases drug efficacy. These block copolymers also increase drug transport in cells forming the blood brain barrier, an important advantage for CNS agents. A similar increase in transport is observed with intestinal cells, which also express P-gp efflux pump, thus facilitating transport of drugs which are administered orally.
The block copolymers also sensitize cancer cells with respect to anticancer drugs. This effect of the copolymers on cytotoxicity can be seen for example with anthracycline antibiotics with respect to drug-sensitive and MDR human breast carcinoma cells. Human Breast Carcinoma MCF-7 cells (ATCC HTB22) and the MDR MCF-7/ADR subline derived from parental cells by selection with doxorubicin (Batist, et al., J. Biol. Chem., 1986, 261, 15544) were maintained in vitro as a monolayer culture in RPMI 1640 media supplemented with 10% fetal bovine serum. The cells were cultured in the drug-free medium for minimum four passages prior to experimental use. The cells then were harvested by trypsinization, plated at 2000 to 3000 cells/well in fresh medium into 96-well microtiter plates, and cultured for 1 to 2 days to allow the reattachment. The doxorubicin with or without copolymers was subsequently added at various concentrations and the cell monolayers were incubated for 2 hours at 37.degree. C. with 5% carbon dioxide. After incubation, the cell monolayers were washed three times with D-PBS and cultured for 4 days in RPMI 1640 supplemented with 10% FBS. The drug cytotoxicity was determined by a standard XTT assay (Scudievo, et al., Cancer Research, 1988, 48, 4827). Briefly, the sterile 1 mg/ml XTT solution in RPMI 1640 containing 5 .mu.l of 1.54 .mu.g/ml phenazine methasulfate solution in sterile PBS was added to the cells (50 .mu.l/well in 200 .mu.l of medium) and incubated for 4 to 16 hours at 37.degree. C. with 5% carbon dioxide. After incubation, the cell monolayers were washed three times with D-PBS. The absorbance at .lambda.420 was determined using a microplate reader. All the experiments were carried out in triplicates. SEM values were less than 10% (P&lt;0.05).The values of IC.sub.50 were determined from the dose response curves.
Experiments were performed in which the IC.sub.50 of free doxorubicin were varied several folds (which is normal for this type of study) to compare the results of these experiments the ratio of IC.sub.50 without and with copolymer was calculated. The results for the following block copolymers are as follows:
 MCF-7 cells MCF-7 ADR IC.sub.50, IC.sub.50, IC.sub.50, IC.sub.50, Composition studied ng/ml ratio ng/ml ratio Doxorubicin in assay buffer 1000 -- 20000 -- Doxorubicin in 0.1% 560 1.78 7200 2.78 Copolymer A Doxorubicin in 0.1% 220 4.54 5800 3.44 Copolymer B Doxorubicin in 0.1% 86 11.6 5000 4.00 Copolymer C Doxorubicin in assay buffer 1000 -- 53000 -- Doxorubicin in 0.02% 950 1.05 22000 2.41 Copolymer D Doxorubicin in assay buffer 850 -- 100000 -- Doxorubicin in 0.0025% 800 1.06 90000 1.11 Copolymer E Doxorubicin in 0.005% 850 1 72000 1.40 Copolymer E Doxorubicin in 0.025% 750 1.13 21000 4.76 Copolymer E Doxorubicin in 0.05% 820 1.04 4000 25 Copolymer E Doxorubicin in assay buffer 1000 -- 18000 -- Doxorubicin in 0.05% T -- T -- Copolymer F Doxorubicin in 0.05% T -- 2500 4.72 Copolymer G Doxorubicin in 0.05% T -- T -- Copolymer H T = formulation toxic at the concentrations used.